BioSpotlight

Chill Out

Patrick C.H. LoKristie Nybo

Pages 51-51

Culture methods for stem cells have come a long way since their introduction in 1998; required growth factors and supplements have been refined and protocols have even been modified for growing cells without feeder layers or serum. But even with these advances, researchers still face significant difficulty preventing spontaneous differentiation of cells: that is, maintaining stem cells in their pluripotent state. Embryonic stem cells (ES) and induced pluripotent stem cells (iPS) are most commonly plated on a layer of inactivated mouse embryonic fibroblasts (MEFs) supplemented with fetal bovine serum and grown at 37°C. This methodology diminishes stem cell differentiation, but does not prevent it; spontaneously differentiated colonies still appear during culture, especially following passaging. Seeking an alternative approach to decrease the numbers of differentiated colonies within their cultures, Belinsky and Antic from the University of Connecticut Health Center (Farmington, CT) modified the standard culture protocol, significantly decreasing differentiation of both ES and iPS cells. Their findings are reported in the current issue of BioTechniques. Recognizing that several cell types, including neural cell precursors and sperm, survive and even thrive when grown at lower temperatures and that differentiation of mybolasts and T-lymphocytes decreases at lower temperatures, the authors decided to simply reduce the incubation temperature for their stem cell cultures from 37°C to 35°C. It turns out that this small modification significantly reduced differentiation rates for both iPS and ES cells in culture. But at what potential cost to cell phenotype? While the growth rates were slowed for cultures maintained at 35°C, as was expected, the authors found no change in the mRNA levels for 3 pluripotency markers or for 2 cold stress markers tested between cells cultured at 35°C and 37°C. Furthermore, the authors demonstrated that colonies maintained at 35°C successfully differentiated into healthy neurons when exposed to the standard differentiation conditions at 37°C. Taken together, these results clearly show that culturing ES and iPS cells at 35°C instead of 37°C significantly reduces differentiation (as well as the labor required to remove differentiated colonies) without altering the pluripotent nature of the cells or their differentiation potential.

A Trip to the Library

Patrick C.H. LoKristie Nybo

Pages 51-51

Massively parallel sequencing (MPS)—i.e., next generation sequencing—has revolutionized molecular biology, catalyzing the explosive growth of genomics and transcriptomics. Construction of a library from target DNA remains an essential step in most MPS protocols. In these cases, proper library preparation and quality control are critical to obtain the best sequence quality as well as to generate the maximum amount of sequence data. Optimizing library construct requires two critical steps: first, the amount of DNA library molecules loaded must be precisely calibrated, as either too little or too much can compromise sequencing data quality or result in failed sequencing runs; and second, the library molecules need to be in an appropriate size range to maximize sequencing throughput. In this month's issue, two papers from different groups describe new methods to optimize MPS library preparation. In the first article, D.J. Park and colleagues at the University of Melbourne (Melbourne, Australia) present a new methodology called Hi-Plex PCR that simplifies targeted MPS resequencing. Several current targeted PCR-based MPS resequencing methods suffer from the need for the labor-intensive normalization of PCR products prior to sequencing in order to obtain libraries of desired size. In contrast, Hi-Plex provides a customizable approach using simple automated primer design software to control the sizes of the PCR amplicons generated in a single PCR reaction from all the targeted regions, which are then size-selected by gel electrophoresis. This flexibility allows Hi-Plex targeted resequencing to be carried out at much less expense and labor than other such techniques. In the second article, J. Bielas and colleagues at the University of Washington (Seattle, WA) describe an accurate and precise method of quality control for MPS libraries using simultaneous quantification and size determination of amplifiable library molecules by droplet digital PCR (ddPCR). Current protocols for MPS library quality control utilize qPCR for quantification, followed by gel or capillary electrophoresis for size determination. But the qPCR and gel selection steps have various drawbacks, such as amplification biases and the need for standard curves. Bielas' group cleverly demonstrate that ddPCR, which had already been validated for absolute DNA quantification, can also be used simultaneously to calculate the size of target DNA due to a linear correlation the authors discovered between the fluorescence amplitude of a ddPCR droplet and the size of the amplicon within it. Their QuantiSize method is not only more accurate than qPCR for quantification, but also conveniently combines the steps for size and concentration determination into a single assay.

Schematic of the single tube, single reaction Hi-Plex PCR mechanism.